Abstract: Recent advances in the genetic reprogramming of mammalian cells are challenging the traditional concepts of cell differentiation and providing new avenues for regenerative medicine. These studies have shown that cellular transcription machinery can be manipulated to reprogram gene expression profiles and redirect cell behavior. Highlighted by recent progress in the development of induced pluripotent stem cells (iPSCs), the principles of genetic reprogramming are also generally applicable to controlling a wide variety of cellular phenotypes. In fact, many cell lineages are defined by a single master regulatory transcription factor, several of which are currently being used to control cell fate for gene- and cell-based therapies and other biotechnological applications. However, current methods for genetic reprogramming are limited by several inefficiencies, including a low frequency of reprogrammed cells, long times to achieve full reprogramming, and insufficient robustness of new cell phenotypes. To address these limitations, we propose to enhance the intrinsic properties of the reprogramming factors through directed molecular evolution. This represents a new conceptual direction in the area of genetic reprogramming and capitalizes on the recent progress in the area of directed evolution for enhancing protein function. Importantly, this approach is broadly applicable to improving the efficacy of any transcriptional regulation machinery and therefore will be beneficial to numerous applications and fields of biomedical research. The outputs of this work will include the discovery of transcription factor variants with enhanced reprogramming capabilities, insights into the biochemistry of morphogenetic transcription factors, and the development of new methods for controlling cell behavior. Additionally, this study will propel the field of directed evolution into the new areas of genetic reprogramming and regenerative medicine. This synergistic incorporation of technologies represents the innovation that will be necessary to translate the transformative advances of biomedical research into real benefits for human health. Public Health Relevance: Scientists are investigating methods to use naturally occurring proteins to coordinate complex cell behaviors, including tissue repair. Although this approach shows tremendous promise, these natural proteins are often insufficient for effectively directing cell activity. This proposal outlines a new and unique technology for evolving engineered versions of these proteins that will be more effective in generating robust cell sources for a variety of applications, including the treatment of cardiovascular disease, neurodegenerative conditions, and musculoskeletal disorders.